Methods for assisting recovery of damaged brain and spinal cord using arrays of X-Ray microplanar beams

ABSTRACT

A method of assisting recovery of an injury site of brain or spinal cord injury includes providing a therapeutic dose of X-ray radiation to the injury site through an array of parallel microplanar beams. The dose at least temporarily removes regeneration inhibitors from the irradiated regions. Substantially unirradiated cells surviving between the microplanar beams migrate to the in-beam irradiated portion and assist in recovery. The dose may be administered in dose fractions over several sessions, separated in time, using angle-variable intersecting microbeam arrays (AVIMA). Additional doses may be administered by varying the orientation of the microplanar beams. The method may be enhanced by injecting stem cells into the injury site.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/054,000, filed on Feb. 10, 2005 now U.S. Pat. No. 7,158,607.

This invention was made with Government support under contract numberDE-AC02-98CH10886, awarded by the U.S. Department of Energy. TheGovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to methods for assistingrecovery of damaged spinal cord and brain and more particularly tomethods of using arrays of x-ray microplanar beams to assist recovery ofdamaged spinal cord and brain.

BACKGROUND OF THE INVENTION

An injury to a portion of the central nervous system (CNS), i.e., thespinal cord or brain, can not be healed by the same means used to treattissue types such as bone, muscle, liver, the peripheral nervous system(PNS), and so on. An injured spinal cord, for example, does not heal andrecover to become functional again, as other tissue types do. Forexample, severed axons at the injury site fail to reestablish synapticconnections, resulting in permanent loss of neural activity.

In addition, beginning over the first two weeks after injury, cellularchanges are triggered that lead to the formation of scar tissue thatacts as a barrier to prevent regeneration. For example, astrocytes, theneuroglial cells which normally provide structural support andprotection to the neurons, transform into reactive astrocytes uponinjury. These reactive astrocytes accumulate to form the bulk of a scartissue that forms, which is referred to as a gliosis (or astrogliosis);and, at a later stage, as a glial scar. This gliotic tissue acts as abarrier to the reconnection of remaining uninjured tissue, includingaxons and neurons, and prevents regeneration of healthy neural tissue.Without regeneration and reconnection, there is no return tofunctionality.

Other processes that are related to the production of reactiveastrocytes and may hamper the recovery are: a) production anddissipation at the injury site of axon-growth inhibiting molecules suchas chondroitin-sulfate proteoglycans (CSPGs) and keratan-sulfateproteoglycans (KSPGs); and b) reaction of the immune system, commonly inthe form of white blood cells (leukocytes) at the entrance to the injurysite.

The barrier formed at the injury site consists of functional barriers orinhibitors, as well as physical barriers. For example, the astrogliosislayer that forms at the injury site, also called the junction (referringto the junction between healthy tissues), prevents the recovery of theset of systems required to restore function including formation of themicrovasculature system. With the failure of early vascular recovery,catastrophic vascular collapse ensues leading to tissue cavitation andstroke-like events. The overall failure of repair of themicrovasculature induces tissue collapse and a failure to bridge thejunction between healthy tissues separated by the glial tissue or glialscar.

One of the only methods currently being researched to solve the problemis irradiation of the injury site with X-rays within two to three weeksafter the occurrence of injury, as described, for example, in Kalderon,et al., “Structural Recovery in Lesioned Adult Mammalian Spinal Cord byX-Irradiation of the Lesion Site,” Proc. Natl. Acad. Sci. USA, Vol. 93,pp. 11179-11184 (October 1996), or in Kalderon, at al., “FractionatedRadiation Facilitates Repair and Functional Motor Recovery after SpinalCord Transection in Rat.” Brain Res. Vol. 904, pp 199-207 (June 2001),both of which are incorporated herein by reference. Although the methodhas produced some encouraging results in laboratory animals, it has notbeen shown to produce substantial functional repair of the spinal cord.

Other methods have focused on limiting the functional expression ofinhibitors or altering the microenvironment that prevents spontaneousregeneration. Yet others attempt to chemically ablate the inhibitoryscar barrier. These methods are limited by the lack of non-invasivemethods for delivery of the chemicals/compounds required to produce theeffect. These methods also exhibit substantial negative “bystander”effects that are overall deleterious to the repair process, oftenexacerbating injury.

There is a need, therefore, for more successful, safe irradiationmethods for assisting functional recovery of a damaged spinal cord orbrain.

SUMMARY OF THE INVENTION

The present invention, which addresses the needs of the prior art,relates to a method for assisting recovery of acute or chronic injury tothe brain or spinal cord by irradiating the injury site with array(s) ofX-ray microplanar beams. The goal of the method of the present inventionis to inhibit the formation of a scar barrier formed by injury to thespinal cord or brain, and simultaneously promote the regeneration of thedamaged microvasculature and glial system to produce substantialfunctional recovery.

The present invention includes a method of assisting recovery of aninjury site of an acute or chronic injury to a brain or spinal cord of asubject. The method includes irradiating the injury site with at leastone array of microbeams. Each array includes at least two parallel,spatially distinct microbeams in an amount and in such a spatialarrangement to allow delivery of a therapeutic dose of X-ray radiationto the injury site.

The therapeutic dose preferably includes an in-beam in-depth dose ineach microbeam substantially in a range from about 30 Gy to about 500Gy.

Preferably, the method includes irradiating the injury site using anumber n of angle-variable intersecting microbeam arrays (“AVIMA”)delivered in different sessions, where each session is separated by atime interval. This method is referred to as “AVIMA with dosefractionation.” After irradiating the injury site with one of theangle-variable intersecting microbeam arrays in one session, theremaining angle-variable intersecting microbeam arrays are preferablygenerated by repeatedly angularly displacing either an X-ray radiationsource generating the arrays or the subject about an axis of rotationthrough a center of the injury site and additionally irradiating anumber (n−1) times, after the time interval required between irradiatingsessions, to generate the number n of angle-variable intersectingmicrobeam arrays. The axis of rotation is parallel to the at least twoparallel, spatially distinct microbeams.

A pattern of radiation is generated such that the angle-variableintersecting arrays intersect substantially only within the injury site.In addition, adjacent angle-variable intersecting microbeam arrays arespatially separated by a displacement angle, which is preferably equalto θ/(n−1), wherein θ is predetermined by an angular access of an X-raysource generating the angle-variable intersecting microbeam arrays tothe injury site.

The method also preferably includes angular displacement of either thesource of the microbeam arrays or the subject by a non-zero integermultiple of the displacement angle between sessions. In other words, anytemporal sequence of the angle-variable intersecting microbeam arraysmay be used to irradiate the injury site.

The total angular spread θ defines the total separation between arrays,and, thus, encompasses the angle-variable intersecting microbeam arrays.In one embodiment, the angular spread is substantially in a range ofabout 130 degrees to about 150 degrees.

The present invention may also include generating a set of nangle-variable intersecting microbeam arrays, where each of theangle-variable intersecting arrays may be generated with the same, or adifferent irradiation orientation of the at least two parallel,spatially distinct microbeams (microplanar beams), where the possibleirradiation orientations are either horizontal, vertical, or slanted(not horizontal or vertical).

In one embodiment, the set of n-angle-variable intersecting microbeamarrays are generated for one irradiation orientation, either horizontal,vertical, or slanted at a particular angle. The method may also includeadditionally generating a second number n of angle-variable intersectingmicrobeam arrays, using a different irradiation orientation of themicrobeams for another n sessions. Each session is separated by the timeinterval. The different orientations may be generated by eitherrepositioning the multislit collimator or changing to a differentmultislit collimator. As a result, the injury site is irradiated for atotal number 2n of sessions. Preferably, the total number 2n of sessionsranges from three (3) to thirty (30) sessions.

The subject may be positioned for irradiation treatment in one of anupright position, a side-reclined, and a slanted position. The arraysare preferably centered around a zero-angle array that impinges on thepatient's back at a 90 degree angle of incidence.

The method of irradiating the injury site with AVIMA may be implementedin sessions separated by the time interval of at least twelve (12) hoursto about seven (7) days.

The array(s) of the present invention preferably include acenter-to-center spacing between adjacent microbeams and a thickness ofeach of the at least two parallel, spatially distinct microbeams,wherein a ratio of the center-to-center spacing to the thickness issubstantially in a range of about 4 to about 16.

In one embodiment, the method includes generating the X-ray radiationwith an X-ray bremsstrahlung source. The microbeams in the array(s)generated with the bremsstrahlung source preferably have a thicknesssubstantially in a range of about 0.1 millimeters (mm) to about 1.0 mm.Alternatively, the method may include generating synchrotron X-rayradiation, in which case, each of the at least two parallel, spatiallydistinct microbeams include a beam thickness substantially in a range ofabout 20 micrometers (μm) to about 100 μm.

In another embodiment, the thickness of the microbeams is substantiallyin a range of from about 0.02 mm (20 μm) to 1.0 mm.

The preferred X-ray radiation from a source generating the microbeamarrays has a filtered broad beam energy spectrum, with a half-powerenergy being substantially in a range from at least about 100 keV toabout 250 keV.

In an additional embodiment of the method of the present invention, themethod further includes delivering stem cells to the injury site.

The method may also include delivering the microbeam array(s) of thepresent invention to the injury site in a plurality of temporallydiscrete pulses of X-ray radiation, which are, in one embodiment,substantially synchronized with a physiomechanical cycle of the subject.Preferably, the physiomechanical cycle includes at least one of acardiac cycle and a cardiopulmonary cycle.

As a result, the present invention provides a method for assistingrecovery of acute or chronic brain injury to the brain or spinal cord.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the cross-section of a microbeamarray of the present invention irradiating an injured spinal cord.

FIG. 2 is a magnified perspective representation of part of themicrobeam array of FIG. 1.

FIG. 3 is a top cross-section view through the injury site of FIG. 1,including a schematic representation of two angularly displacedexposures of the injured spinal cord with the microbeam array inaccordance with an embodiment of a method of the present invention.

FIG. 4 is a top cross-section view through the injury site of FIG. 1,including a schematic representation of three angularly displacedexposures of the injured spinal cord with the microbeam array inaccordance with an embodiment of a method of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of assisting physiological,neurological, and functional recovery of a damaged spinal cord or brainby delivering a therapeutic dose of X-ray radiation to the injury siteusing microplanar X-ray beams. The method preferably inhibits orminimizes the formation of an astrogliotic or gliosis barrier at thesite of injury and simultaneously promotes the regeneration andreconnection of axons. The in-beam therapeutic dose at the injury site,delivered by the microbeams, preferably operates to promote a lesshostile environment in which the presence of growth inhibiting moleculesand leukocytes is minimized and the recovery of the glial system andremyelination processes are encouraged.

Referring to FIG. 1, the present invention provides a method ofassisting functional recovery of a damaged spinal cord or brain byirradiating an entire area of spinal cord or brain damage, i.e., theinjury site 10, with at least one array of microplanar beams 12 of X-rayradiation. The particular pattern of radiation formed by the microbeamirradiation at the injury site 10 preferably both inhibits the formationof a gliotic barrier formed by injury to the spinal cord or brain, andpromotes the regeneration of the damaged microvasculature general systemto produce substantial functional recovery. The method of the presentinvention preferably promotes this regeneration by encouraging abridging of healthy tissue on opposite sides of the damaged area. Thisis accomplished preferably by reducing the concentration of reactiveastrocytes and axon-growth inhibiting molecules such ascondroitin-sulfate proteoglycans (CSPGs) and keratan-sulfateproteoglycans (KSPGs) and immune-response cells such as leukocytes, andpromoting remyelination to produce substantial physiological,neurological, and functional recovery.

The use of microbeam arrays is known in the prior art for use inmicrobeam radiation therapy (MRT) as an experimental method for thetreatment and ablation of tumors, as described, for example, in U.S.Pat. No. 5,339,347 to Slatkin et al., which is incorporated herein byreference. MRT differs from conventional radiation therapy by employingarrays of preferably parallel and planar microbeams of radiation(microplanar beams). The thickness of each microplanar beam is at leastone order of magnitude smaller in thickness (or diameter if cylindricalrather than planar beams are used) than the smallest radiation beams inconventional clinical use. The entire width of the array, however, maystill cover an area comparable to that covered using broad beam. Theadvantage over conventional broad beam radiation for tumor ablation andcontrol is that the irradiated normal tissue in the path of theindividual microbeams, which is often irreversibly damaged byconventional radiation therapy, is allowed to recover from any radiationinjury by regeneration from the supportive cells surviving between themicrobeams.

The Slatkin et al. patent discloses the segmentation of a broad beam ofhigh energy X-ray into arrays of parallel microbeams (beams of thicknessless than about 1 millimeter (mm)), and a method of using the microbeamsto perform radiation therapy on tumors. The tumor receives a summedabsorbed dose of radiation exceeding a maximum absorbed dose tolerableby the target tissue by crossing or intersecting microbeams at thetarget tissue. The irradiated in-beam normal tissue is exposed only tonon-crossing beams. Normal tissue between the microbeams receives asummed absorbed dose of radiation, called a valley dose, less than themaximum tolerable dose; i.e., normal tissue in the valleys receives anonlethal dose, leaving surviving supporting cells. The surviving cellsmigrate to the irradiated normal tissue in the path of the microbeam,allowing normal tissue in the path of the beam to recover from radiationinjury.

The method of the present invention includes irradiating the injury siteof an acute (injury site less than 20 days old) or chronic (more than 20days old) injury to the spinal cord or brain with an array ofmicrobeams, which are preferably parallel microplanar beams, instead ofusing a conventional, unsegmented broad beam, to assist recovery fromthe injury. The use of microbeam arrays for recovery of damaged spinalcord and brain takes advantage of the normal tissue-sparingcharacteristics offered by the geometry of microbeam arrays, describedsupra. The microbeam arrays allow the normal tissue, including that ofthe central nervous system (CNS) which encompasses the spinal cord, torecover almost completely from the damage produced by the radiation.

In addition, it is believed that irradiation of the injury site withmicrobeam arrays advantageously assists in recovering the capillaryblood vessels that are injured in a reversible manner. Segments of theendothelial cells in the direct path of the microbeams are destroyed (aswould occur with conventional broad beams), but are regenerated byendothelial cells and vessel wall cells surviving between microbeams.Consequently, the massive vascular collapse and tissue collapse thatoccurs with conventional broad-beam treatment of spinal cord and braininjuries is avoided, and the tissue's microstructure, which is mainlythe capillary blood vessels, is spared.

Specifically, in the prior art broad-beam methods for treating brain andspinal cord injury, the width of a conventional broad beam is too largeto allow the above recovery process to proceed. Because the capillaryblood vessels constitute the basic infrastructure of the tissue, itssurvival is the most important factor in the recovery of the entire CNStissue from microbeam arrays.

The method of the present invention is also believed to advantageouslypromote the restoration of the “in-beam” glial cells and myelin in thehealthy CNS tissue just outside the injury site, which are damaged bydirect irradiation as the microbeams traverse a path to the injury site.For example, although the microbeams of the present invention can killprogenitor and mature glial cells and destroy myelin in the path of theindividual beams, this system component also recovers, this time fromthe endogenous progenitor glial cells in the CNS tissue which survivebetween microbeams. These surviving cells migrate to the neighboringareas of direct exposure to microbeams in which the tissue has beendepleted of glial cells. The cells then differentiate and become matureand functioning glial cells, including mature and myelinatingoligodendrocytes. Finally, a remyelination process begins whereby lostmyelin is replaced with a new, functioning myelin.

The microbeam array essentially “cleans” the gliosis produced at theinjury site by reactive astrocytes, which forms the junction betweenhealthy tissue, by using an adequately high dose, or “therapeutic dose”of X-ray radiation. The therapeutic dose as used herein is the in-beam,in-depth (at the depth of the injury) radiation dose, typically measuredin units of Gray (“Gy”), required at the injury site to allow thedamaged spinal cord or brain to recover from the injury. The therapeuticdose must, however, remain below the threshold for inducing permanentradiation damage to normal tissue in the beam path.

Preferably, the therapeutic dose of irradiation kills the glial cells inthe path of the microbeams, mostly reactive astrocytes whose aggregationwill (or has) produced the gliotic barrier, without damaging the corditself. It is believed that the net effect is that the microvasculatureat the junction reestablishes itself and the bridging process at thejunction proceeds, leading to the rejoining of axons, functionalremyelination and, consequently, functional restoration within thedamaged spinal cord or brain. Basically, the microbeams are used toclean an injured area temporarily of inhibitors of regeneration byridding the zone of reactive astrocytes, leukocytes and axon-growthinhibiting molecules such as CSPGs and KSPGs. Previous methods ofbroad-beam irradiation required low tolerable doses of irradiation toprevent radiation damage to normal tissue. These low doses are notcapable of substantially assisting recovery of the injury site by, forexample, cleaning an area entirely of reactive astrocytes, leukocytesand CSPGs/KSPGs.

The therapeutic dose, therefore, is preferably the dose required tosubstantially cleanse or remove the regeneration inhibitors, e.g., thereactive astrocytes, CSPGs, and KSPGs, at least temporarily, from theirradiated in-beam portion of the injury site.

Referring again to FIG. 1, in the method of the present invention, aninjury site 10 of a portion of a nervous system, for example, the spinalcord 14, of a patient is irradiated through surrounding bone and tissue15 with at least one array of microbeams 12. The array 12 includes atleast two parallel, spatially distinct microbeams 16. The number andspatial arrangement of the microbeams in the array 12 are so chosen todeliver a therapeutic dose of X-ray radiation to the injury site 10through the microbeams 16.

As indicated in FIG. 1, the injury site 10 as used herein refers to avolume targeted for irradiation treatment, which encompasses the tissueaffected by the acute or chronic injury in addition to a marginal volumearound the affected or damaged tissue. The marginal volume is determinedby factors known to those skilled in the art of conventional radiationtreatment. Such factors include the accuracy of the radiation sourceused in hitting a designated volume, and considerations of possiblespreading or misestimation of the extent of tissue affected by the acuteor chronic injury.

As shown in FIG. 2, the microbeams 16 in an array 12 preferably includeparallel irradiation planes (vertically oriented in the embodiment shownin FIGS. 1-4, but could also be horizontal or slanted) 18, and may alsobe referred to as microplanar beams 16. These microplanar beams 16preferably have a substantially rectangular cross-section, with a beamthickness 20 corresponding to the short side of the rectangularcross-section. The array 12 is also characterized by a center-to-centerbeam spacing 22 and a width 24.

The term “beam spacing” as used herein refers to the center-to-centerbeam spacing 22, which is generally measured as the distance between themaximum intensity profile points of adjacent microbeams. In the case ofa flat intensity dose profile in each microbeam, the center-to-centerbeam spacing 22 would be measured as the distance between the mid-pointsof the intensity profiles of adjacent microbeams.

In addition, the microbeams 16 generated in the array 12 are preferablysubstantially collimated at least in the plane perpendicular to theradiation plane, so that the spacing 22 is substantially maintainedbetween the beams 16 as they traverse the subject. The microbeams 16preferably also include substantially sharp, well-defined edgesseparating adjacent microbeams in the array 14, for sharp dose fall-off,thus minimizing the valley dose between the beams 16.

Preferably, the width 24 of the microbeams and a total length 26 of thearray 12 are large enough to irradiate the entire volume enclosing theinjury site 10, which includes some additional margin. A typical size ofthe microbeam array may be about 4 cm wide across a vertical spinalcolumn, e.g., and 6 cm high along the vertical spinal column.

If the injury site 10, represented by a circle around a narrowhorizontal line representing the location of a center of the injuredtissue in FIG. 1, covers a height greater than that (width 24) of themicrobeam array, however, then the therapeutic dose may be provided overthe entire height of the injury site by translating the patient in thepath of the at least two microbeams of the microbeam array (vertically,in FIG. 1). The method may include using a stepwise scanning method(step-and-shoot) in which the subject or source is translated in thebeam at a certain step height after each irradiation step or in acontinuously scanning method in which the subject or source istranslated continuously. Similarly, if the extent 26 of the array 12does not cover the injury site 10, arrays may be generated side-by-sideusing a stepwise scanning method.

Although the microplanar beam geometry described supra is preferred, anymicrobeam and microbeam array geometry capable of safely and effectivelydelivering the therapeutic dose and assisting recovery of the injurysite are within the scope of the invention. For example, an array ofcylindrical pencil beams may be used. In addition, the array may be atwo-dimensional array, for example, a rectangular array of microbeams,the microbeams of the array being preferably substantially equallyspaced. A rectangular or other two-dimensional array may be preferredwhen the cross-sections of the microbeams are circular, square, orotherwise substantially radially symmetrical.

Preferably, the microplanar beams 16 of the array 12 of the presentinvention are produced simultaneously using a multislit collimatorhaving any of various designs known in the art. Such collimators havemultiple radiation transmissive apertures that allow an array ofspatially distinct microbeams to be simultaneously produced from asingle wide radiation beam.

The multislit collimator generating the array 12 is preferablypositioned in front of the X-ray source, but very close to the subject'sbody, in order to minimize the so-called “penumbra” or shadow of thesource beam. Placing the collimator close to the beam, therefore, helpsmaintain the desirable sharp edges of the beams 16 throughout thesubject.

The single X-ray radiation beam impinging on the multislit collimatormay be generated by any source of X-rays capable of producing therequired therapeutic dose. For example, the appropriate X-ray radiationmay be generated by filtering radiation produced by a high energysynchrotron or an X-ray tube (bremsstrahlung radiation). The fluencerate of the source used to implement the method of the present inventionis preferably high, so that exposure times are sufficiently short,reducing the possibility of smearing the microbeam dose pattern producedat the injury site.

One possible source of X-rays is a wiggler insertion device in aso-called “beamline” of a high-energy electron storage ring of an X-raysynchrotron. An exemplary beam source is the superconducting wigglerinsertion device of the X17B beamline of the National Synchrotron LightSource at Brookhaven National Laboratory, Upton, N.Y. A conventional“planar” wiggler uses periodic transverse magnetic fields to produce abeam of rectangular cross-section, typically having a horizontal tovertical beam opening angle ratio on the order of 50:1. In analternative embodiment, the radiation beam is obtained from a “helical”wiggler, a configuration capable of producing a substantially lessanisotropic beam.

In a preferred embodiment, the source will be a bremsstrahlung X-raygenerator. The bremsstrahlung X-ray source may include a high-throughputrotating anode X-ray tube operating at a very high voltage (preferablyabout 150 kV-peak or higher) and a very high current (100 mA or higher).

Whether the source is a bremsstrahlung source or storage ring, the beamis preferably filtered with copper or heavier elements to eliminate thelow end of the energy spectrum, thus producing a higher mean spectralenergy. Preferably, the half-power energy of the filtered X-ray beam isin the range of 100-250 keV. For example, to produce the desired energyspectrum from a high-throughput rotating anode tube operating at 140-250kVp, the beam should be heavily filtered, e.g., through about 1-10 mmthick copper.

Most preferably, the X-ray source is one or more X-ray tubes generatingbremsstrahlung radiation. One X-ray tube may be used and either thesubject or the tube repositioned to produce the microbeam arrays of thepresent invention. Alternatively, multiple X-ray tubes may be maintainedin fixed positions arranged around the patient, at the appropriateangles of incidence to the patient, to generate the arrays in accordancewith the methods of the present invention.

The focal spot size of the X-ray tube(s) should be minimized to reducethe beam penumbra, assuring sharp edges and thus a sharp dose fall-offto minimize the valley dose. Optimally, X-ray tubes for use with themethod of the present invention include a) a source spot size smaller orcomparable to the thickness of the microplanar beams, b) a stable standto keep the tube fixed at different angles with little vibrations, andc) a geometry that allows placement of the tube close to the subject(preferably within 50 centimeters), in order to maximize the dose rate.

Referring again to FIG. 2, the optimal beam thickness 20 and spacing 22will vary depending on the characteristics of the source. The ratio ofbeam thickness 20 to center-to-center beam spacing 22, however, willpreferably be in a range of from about 1 to 4 to about 1 to 16,regardless of whether the source is a high-energy electron storage ringor a bremsstrahlung X-ray tube.

In one embodiment, the ratio of thickness 20 to center-to-center beamspacing 22 is in a range from about 1 to 6 to about 1 to 8.

In another embodiment, the ratio of beam thickness 20 tocenter-to-center beam spacing 22 will be substantially equal to 1 to 7.

If a synchrotron source is used to generate the microbeams and implementthe method of the present invention, for example, one preferable choiceof beam thickness 20 and spacing 22 are 30 micrometers (μm) and 210 μm,respectively (1:7). If a bremsstrahlung X-ray tube is used, a morepreferable choice is a beam thickness 20 of about 0.7 mm (or within arange of about 0.5 mm to 0.9 mm) and beam spacing of about 4.9 mm (orwithin a range of about 3.5 mm to 6.3 mm). Though a larger beam size ispreferred when using an X-ray tube, the same ratio of thickness 20 tospacing 22 of 1 to 7 as the synchrotron-beam example is preferred.

The preferred thickness of the microbeams for implementing the method ofthe present invention using a bremsstrahlung source is larger, becausethe effective source spot size in bremsstrahlung sources is larger thanthat of a storage ring. The effective source spot size is defined as theangle at which the source spot size is viewed from the position of thetarget, or, equivalently, the ratio of the source size to the distancebetween the source and the target. Although the actual spot sizes in thesynchrotron and the bremsstrahlung sources might be comparable to eachother, the source-to-target distance is much larger for the synchrotronsource (about 10 m) than for the bremsstrahlung source (about 1 m).

In a preferred embodiment, therefore, the method includes providing themicrobeam array of X-ray radiation using a bremsstrahlung radiationsource, e.g., X-ray tubes, where the thickness of the microbeams issubstantially in a range of about 0.1 mm to 1.0 mm. In addition, theratio of beam thickness 20 to center-to-center beam spacing 22 ispreferably substantially equal to 1 to 7.

In another embodiment, the beam thickness 20 is substantially equal toor greater than about 0.02 mm (20 μm) and less than or equal to about1.0 mm.

In still another embodiment, preferably using synchrotron X-rayradiation, the beam thickness is substantially equal to or greater thanabout 20 μm and less than or equal to about 100 μm.

In yet another embodiment, microbeams are provided which include a widthsubstantially less than or equal to about one millimeter.

The therapeutic dose of the present invention to allow substantialrecovery of the brain or spinal cord from the injury is preferablywithin a range of about 30 to about 500 Gy in-beam in-depth dose, wherethe in-beam in-depth dose refers to the dose inside each microplanarbeam at the depth, within the body, of the spinal cord or brain.

It is well known to those skilled in the art that the threshold dose, ormaximum tolerable dose before neurological and other complications ofradiotherapy arise, increases as irradiated volumes of tissue are madesmaller. Equivalently, as the irradiated volume (beam thickness)increases, the maximum tolerable dose decreases. The beam thickness,therefore, partially sets the upper limit of the therapeutic dose thatcan be safely administered.

In the preferred embodiment of the present method, a bremsstrahlungsource is used to deliver the therapeutic dose. As discussed supra, thethickness of the microbeams produced will then preferably be in a rangeof about 0.5 mm to about 0.7 mm with a ratio thickness to beam spacingof about 1:7. The therapeutic dose for this case is then limited to asmaller upper limit. In one embodiment, the in-beam, in-depth dose ispreferably within a range of about 30 to about 200 Gy.

The required rate at which the dose is delivered, i.e., the dose rate,depends on the dose to be administered. If the in-beam dose required isabout 100 Gy, then the dose rate should be at least about 2 Gy perminute to allow administration of the 100 Gy dose in about 50 minutes.Both types of sources are capable of this dose rate. In general,however, a synchrotron source can provide a much higher dose rate thanX-ray tubes.

The injury site may be irradiated, according to the present invention,within 20 days or less after the injury occurred, i.e., while the injuryis still classified as an acute injury

The injury site may also be irradiated 20 days after the injury toassist recovery of a chronic injury.

In addition, the therapeutic dose may be delivered either in a singlesession (single dose fraction) or in several sessions in so-called “dosefractions” or “dose fractionations” separated by a time interval. Asreferred to herein, however, the dose fractions are defined in the sameway as the therapeutic dose: each dose fraction includes the in-beamin-depth therapeutic dose required at the injury site to allow thedamaged spinal cord or brain to substantially recover from the injury.

In other words, the transient damage to the irradiated portion of theinjury site is sufficient, in every session, to allow that portion to beat least temporarily cleaned of regeneration inhibitors, so thatneighboring progenitor glial cells can migrate to the injured portionand begin a process of repair.

The in-beam tissue dose (i.e., in-depth dose) in all fractions must behigh enough to ablate the target cells, such as progenitor glial cellsor reactive astrocytes; i.e., the same as the in-beam in-depththerapeutic dose. Therefore, no matter how many dose fractions are used,each dose fraction must be above the cell-ablation threshold, and is notaccumulative. In this sense, the term “dose fraction” is simply thein-beam in-depth therapeutic dose required, and not a fraction thereof.However, the “valley” dose, which is accumulative, must be kept as lowas possible (through the optimal choice of the beam width, beam spacing,and the in-beam dose). Here the limitation is that the accumulativevalley dose for the entire treatment period will not surpass the limitof tissue tolerance for dose-fractionated broad beams in the samefractionation schedule.

In a preferred embodiment, several dose fractions, i.e., therapeuticdoses, are administered to the subject by irradiating the injury site insubsequent sessions from different angles with so-called “angle-variableintersecting microbeam arrays (“AVIMA”).

By applying dose fractionation with AVIMA, normal tissue in the path ofthe beam is irradiated only once, thus minimizing possible radiationdamage to normal tissue. In addition, regions of the injury sitereceiving multiple in-beam in-depth therapeutic doses, receive a highersummed dose to more effectively clean the gliotic tissue. Anotheradvantage of the AVIMA geometry, as described below, is that a greaterarea of the injury site is irradiated.

Referring again to FIG. 3, in an embodiment of a method of the presentinvention, therefore, a therapeutic dose is administered in more thanone session using angle-variable intersecting microbeam arrays. Themicrobeam arrays intersect substantially only at the injury site 10, asindicated by the grid-like regions of intersection 28 in FIG. 3 withinthe area of the injury site 10. The method using AVIMA preferablyincludes irradiating the injury site 10 with arrays of angle-variablemicrobeams centered around and including a “zero angle” array 30.Preferably, the zero angle array 30 is directed to squarely impinge onthe subject's back, i.e., the array 30 is normally incident (90 degreeangle of incidence) on the back. Adjacent angle-variable intersectingarrays (34, 30) are separated by an angular displacement 32. A firsttherapeutic dose is delivered in one of the angle-variable microbeamarrays 30 at “Time Zero” on Day 1 of treatment during a first session.

The method further includes angularly displacing by a non-zero integermultiple of the displacement angle 32 either the X-ray source or thesubject about an axis of rotation 35 through a center of the injury site10 (See FIG. 1) and, after a time interval between sessions,additionally irradiating the injury site 10 with one of the angularlydisplaced, i.e., angle-variable intersecting microbeam arrays 34. Theaxis of rotation 35 is preferably substantially parallel to theirradiation planes 18 of the microbeams 16 (see FIGS. 1-2). The methodincludes repeatedly angularly displacing and additionally irradiatingafter the time interval, until the injury site 10 has been irradiated atotal number n of times over n sessions separated by the time interval,by a total number n of AVIMAs.

In another embodiment, the number of irradiations may also be doubled byreirradiating a second time at each angular position of eachangle-variable array using a second irradiation geometry. The secondirradiation geometry is provided by either reorienting the multislitcollimator provided between the source and the subject, or by changingto a different collimator. The two different positions may includedifferent slanted orientations (not horizontal or vertical), horizontal,or vertical orientation. The method described supra of administeringangle-variable intersecting arrays, wherein each array comprises atherapeutic dose of radiation would then be repeated. When theirradiation geometry is described by vertically-oriented microbeams, thesubject or source is rotated by some multiple of the angulardisplacement angle 32 from one fraction to the next (for differentAVIMAs) about a vertical axis centered through the injury site. When theirradiation geometry is described by horizontally-oriented microbeams,the subject or source is rotated about a horizontal axis. When slantedmicrobeams are used, the angle of rotation or displacement is parallelto the slanted axis. Though a temporal sequence of sessions mayalternate randomly in both irradiation geometry (orientation of theirradiation planes) and angular displacement, it is probably morepractical to first irradiate the injury site at all angles of the AVIMAsusing one irradiation geometry before turning/changing the collimator(or subject) to a second irradiation geometry.

In one embodiment, every angle-variable intersecting microbeam arrayirradiating the injury site can have a different irradiationorientation.

The following further describes the most likely geometries of theirradiations, although not the only ones, given the practicality ofimplementing the present method with existing X-ray sources. Themicrobeam arrays may be propagated horizontally from a source, whichwould be the case for most synchrotron beams and some X-ray tubes.Optionally, the arrays may be propagated vertically from a sourcepointing up from the floor or down from the ceiling (most X-ray tubesbeam).

Horizontal beams will preferably be administered with the patient eithersitting upright on a patient positioning chair, or lying down on his/herside on a patient positioning bed. Vertical beams will preferably beused with the patient lying down on the bed other on his/her back (withthe beam coming up from the floor), or lying down on the bad on his/herfront (with the beam coming down from the ceiling). For both thehorizontally and vertically propagating beams, the patient's back istoward the beam and the angle of incidence of the microbeams in thearray is 90 degrees for the “zero angle” of irradiation. The additionalarrays are preferably centered around the zero angle array. In thediscussion that follows the “Time Zero” or first dose fraction is at thezero angle. Subsequent irradiation angles may vary widely for differentdose-fraction administration. The parallel irradiation planes of theindividual microbeams in an array are preferably aligned eithervertically or horizontally, at least in the geometry of “zero angle.”However, the alignment angle may be titled for other irradiationgeometries as discussed above, by repositioning the subject or thecollimator in front of the X-ray source generating the arrays. In otherwords, the irradiation geometry may be different for every dose fractionadministration for every angle-variable intersecting microbeam array.

Referring to FIGS. 1-4, the following example describes theangle-changing geometry. In this example, the beam array 12 propagateshorizontally, the X-ray source irradiating the injury site 10 from theside. The microplanar beams 16 in the array are vertical, and thepatient sits upright (spinal cord 14 vertical) with the back to the beamfor the zero angle geometry (first fraction). The incident angle of thebeam 12 on the subject between the dose fractions will then changelaterally, as shown in FIG. 3. In other words, either the source or thesubject rotates around a vertical axis that goes through the center ofthe injury site 10 in the patient's spinal cord 14 between dosefractions.

AVIMA with dose fractionation may be implemented similarly in anycombination/orientation of the beam and patient geometries. For example,in the above example (horizontally propagating beam; upright oron-the-side patient) one can also use horizontally oriented microplanarbeams instead of vertically oriented ones (in the zero angle geometry),and rotate the source or the patient about a horizontal axis that goesthrough the injury site (although the beam can be rotated only whenproduced by an X-ray tube).

Not all combinations of the motions, however, are possible. For example,for vertically propagating beams (patient lying on his/her back orfront) there will be two possible orientations of the parallelirradiation planes 18 of the microplanar beams 16: either parallel tothe spinal cord or perpendicular to it. If parallel, then the source orpatient should be rotated about the spinal cord between dose fractions,while, if perpendicular, the rotation should be about a horizontal axisperpendicular to the spinal cord. In both these cases it will not beeasy to rotate the patient because his/her general positioning maychange, thus the rotation is preferably implemented by rotating thesource.

Several dose fractions (sessions), may be administered using AVIMA,preferably ranging from a total of about 3-30, using one or more of theirradiation geometries of the microplanar beams described above. Forexample, in the example using a horizontally propagating beam andpatient sitting upright or lying on his/her side (side-reclinedposition), the therapeutic dose can be given at each angle correspondingto the AVIMAs once with horizontally oriented microplanar beams and oncewith vertically oriented ones. Each session is separated by a minimumtime interval.

In one embodiment, the minimal time between sessions in which each dosefraction is administered is at least about six (6) hours.

In another embodiment, the time between sessions preferably ranges fromabout twelve (12) hours to about thirty-six (36) hours.

In yet another embodiment, the time between sessions preferably rangesfrom about twelve (12) hours to about seven (7) days.

Retreatment of chronic spinal cord injury is also within the scope ofthe method of the present invention. The entire treatment is preferablyrepeated, if needed, within about six (6) to eighteen (18) months aftera previous treatment.

A further illustration of the AVIMA method of the present inventionincludes the preferred geometry of the microbeam arrays of the presentinvention, for which the beam thickness is about 1/7 of thecenter-to-center beam spacing. In each dose fraction, therefore, about14.3% of the target's volume is irradiated. If the dose is adequatelyhigh to kill all progenitor glial cells residing in the direct beampath, 14.3% of the entire population of such cells are ablated in thefirst dose faction administration. The additional percentage ofprogenitor cells in subsequent fractions, preferably administered atwide angles to the direction of the first or “zero angle” beam, isslightly smaller than this number, due to the geometry. The microbeamsof the angularly displaced arrays will intersect a subregion of the zeroangle-irradiated regions of the injury site. In other words, smallerareas of the array from one dose fraction will cross those from anearlier fraction. In general, if the letter “r” denotes the ratio ofbeam width divided by beam spacing, and letter “n” denotes the number ofdose fractions, the surviving fraction “s” of the directly-hitprogenitor glial cells after n dose fractions is defined by equation (1)to be:s=1−(1−r)^(n)  (1)

Therefore, for r=0.143 (1:7 thickness to spacing ratio) and n=5 dosefractions (sessions), one finds s=0.54, i.e., about 54% of the cellswill be hit directly at least once. If each directly-hit progenitorleads to a rejuvenated, mature glial cell, then about 54% of the glialsystem will be rejuvenated in this example.

If two sets of irradiations are carried out using two differentirradiation geometries, at each of n angular positions of theangle-variable intersecting microbeam arrays, then 2n dose fractions areadministered over 2n sessions, and s=1−(1−r)^(2n).

Referring to FIG. 4, in dose-fraction with AVIMA, the administrationangles for different dose fractions are chosen to provide the widestpossible angular spread θ 36 among the fractions. The angular spread 36encompasses all of the angle-variable intersecting microbeam arrays. Themaximum spread 36 is limited to the angular access afforded by thelocation of the injury, patient-positioning ability of the equipment andthe irradiation source. Because the irradiation is administered to theback, less than 180° of angular spread will be available. A reasonablevariation is probably about 140° angular spread θ36 for each possibleangle variation geometry, i.e., from zero angle in which the patient'sback is precisely facing to beam, to ±70°.

In one embodiment, the angular spread 36 is substantially in a rangebetween about 130 degrees to about 150 degrees.

In general, the formula to choose the angular spacing between the beamsfor n dose fractions or sessions is preferably equal to θ/(n−1). Forexample, for n=3 (three fractions only), and θ=140°, the angulardisplacement will be 70° between irradiations, and the angles will be−70°, 0°, and +70°, where 0° represents the angle at which the beams hitthe patient's back. Similarly, for n=5, the microbeam array is angularlydisplaced by about 35° between irradiations; thus the angles will be−70°, −35°, 0°, +35°, and +70°. As indicated supra, these dose fractionscan also be divided into two groups per session, using differentirradiation geometries or orientations of the microplanar beams, thusincreasing further the angular range between fractions.

The choice of the orientation of the microplanar beams with respect tothe spinal cord may have physiological/neurological consequences for tworeasons. First, the spinal cord is essentially made of long axons eithercoming from the brain or returning to the brain. Second, the breathingcycle of the patient may cause the spinal cord to move up and down orsideways, a motion which may smear the dose distribution of themicrobeam array.

In general, it may be safer to have the microplanar beams not parallelto the cord because they may engulf long segments of axons. Thisconsideration might be more important when used wider microbeams (e.g.,0.7 mm wide). The breathing motion should also be taken into account,however, which is more important for microbeam arrays with small beamwidth and small beam spacing. For example, for a horizontallypropagating beam with the patient sitting upright with his/her back tothe beam, it might be advantageous to use horizontally orientedmicroplanar beams than vertical ones not to be parallel to the cord,although in this geometry an array with horizontal microplanar beams maybe more vulnerable to beam smearing because the breathing motion may bemore an up-and-down one.

Preferably, the method of the present invention includes maximizingremoval of the substantial amount of regeneration inhibitors from theportion of the injury site by optimizing several factors, such as thetherapeutic dose of a single fraction, and minimizing the valley dose tominimize damage to the adjacent tissue between the microbeams. Thein-beam in-depth therapeutic dose to valley dose ratio may be alsooptimized by controlling at least one of the thickness of themicrobeams, the spacing between microbeams, the ratio of the thicknessto the spacing, and the energy spectrum of the microbeam array.

In order to optimize the in-beam therapeutic dose to valley dose or“peak-to-valley” ratio, the dose fall-off at the edge of any individualmicrobeam inside a microbeam array of the present invention ispreferably sharp enough at beam energies of between about 50 keV andabout 200 keV, and at tissue depths of from about 1 cm to about 40 cm,to result in large peak-to-valley dose ratios for the present invention.Such large ratios allow dose planning so that the peak dose will belethal to the regeneration inhibitors such as reactive astrocytes andaxon-growth inhibiting molecules of CSPGs and/or KSPGs, while valleydoses will be low enough to allow most normal and regenerative cells,such as progenitor glial cells, to survive the radiation. These glialcells can then migrate to the cleansed irradiated portion of the injuredsite to assist in tissue repair.

The appropriate selection of the parameters of microbeam fieldconfiguration and peak dose is critical to the efficacy of microbeamradiation therapy for assisting recovery of damaged nervous system suchas the brain and spine. The peak dose along the microbeam axis and thecenter-to-center spacings of the microbeams must be appropriatelyselected to ensure sufficiently low doses to tissue present in thevalleys between the microbeams. The implemented combination of dose andconfiguration allows endothelial cells, oligodendrocytes (in braintissue), and glial cells between the microbeams to divide and torepopulate the irradiated portion of the injured site, as well as thein-beam tissues damaged by the radiation treatment outside the injurysite. Thus, unidirectional exposure does not permanently damage normaltissue.

In one embodiment, a therapeutic in-beam in-depth dose is delivered infractionated doses in a range from about 30 Gy to about 500 Gy. Thenumber of fractionated doses is preferably in a range of about 3 to 30doses.

The success of the AVIMA methods of the present invention relies inpart, therefore, on maintaining accurate microbeam placement fromsession to session, and during the exposure time of one session.Therefore, the methods of the present invention are preferablyimplemented using stereotactic apparatus to preferably eliminate or atleast substantially minimize so-called macromotion, for example, fromrandom movements of the patient.

Other sources of motion, referred to as tissue “micromotion” asdiscussed supra are induced by physiomechanical cycles, namely, cardiacpulsations and pulmonary or respiratory cycles. The methods of thepresent invention may, therefore, be performed in a pulsed mode, thepulsations of the microbeams being synchronized with the subsequentrhythmic displacements of the injury site. Most preferably, the pulsesare synchronized with an electrocardiogram, as described, for example,in the Slatkin, et al. patent.

In one embodiment of the present invention, the high energyelectromagnetic radiation source is pulsed, so that the microbeam arrayis delivered in a plurality of temporally discrete pulses. Preferably,the temporally discrete pulses are substantially synchronized with aphysiomechanical cycle of the patient. Most preferably, thephysiomechanical cycle includes at least one of a cardiac cycle and acardiopulmonary cycle.

In another embodiment of the present invention, a method of the presentinvention includes combining the microbeam irradiation of the injurysite using any of the beam geometries and characteristics discussedherein with transplantation of embryonic stem (ES) cells to the injurysite. It is believed that the combination of these two methods willgreatly enhance the recovery of an injury to a portion of a nervoussystem, particularly, to the brain or spinal cord.

It is very much likely that the population of endogenous glial stemcells naturally existing in the spinal cord (also called progenitorglial cells), once stimulated by microbeam irradiations to proliferateand differentiate, will be adequate to complete the process of the“glial system rejuvenation” which may be necessary to allow axonalgrowth and reconnection, as well as remyelination toward the recoveryfrom spinal cord injury. However, it is also possible that externaladministration of stem cell will assist the recovery process byaccelerating it or by making it more efficient. The administered stemcells might be human or animal embryonic cells. The stem celladministration may or may not require a surgical process.

The methods of the present invention may be used to treat both acutecases of spinal cord or brain injuries, existing for up to 20 days afterinjury, and chronic cases of spinal cord injuries, which are older than20 days.

Although illustrative embodiments of the present invention have beendescribed herein with reference to the accompanying drawings, it is tobe understood that the invention is not limited to those preciseembodiments, and that various other changes and modifications may beeffected therein by one skilled in the art without departing from thescope or spirit of the invention.

1. A method of assisting recovery of an injury site of an acute orchronic injury to a brain or spinal cord of a subject, the methodcomprising: irradiating the injury site with at least one array ofmicrobeams comprising at least two parallel, spatially distinctmicrobeams in an amount and spatially arranged to deliver a therapeuticdose of X-ray radiation to said injury site, said therapeutic dose ofX-ray radiation inhibiting the formation of a scar barrier andsimultaneously promoting the regeneration of a microvascular and glialsystem at said injury site.
 2. The method of claim 1, wherein saidirradiating further comprises delivering the therapeutic dose with theat least one array of microbeams to the injury site repeatedly in anumber n of sessions, each session being separated by a time interval.3. The method of claim 2, wherein the at least one array comprises anumber n of angle-variable intersecting microbeam arrays, the methodfurther comprising generating the angle-variable intersecting microbeamarrays.
 4. The method of claim 3, wherein said generating comprises thesteps of: irradiating the injury site with one of the angle-variableintersecting microbeam arrays in one session; angularly displacing atleast one of an X-ray radiation source generating the at least one arrayand the subject about an axis of rotation through a center of the injurysite, wherein the axis of rotation is parallel to the at least twoparallel, spatially distinct microbeams, to produce a second one of theangle-variable intersecting microbeam arrays; additionally irradiatingthe injury site with the second one of the angle-variable intersectingmicrobeam arrays after the time interval in a second session; andrepeating said angularly displacing and additionally irradiating anumber (n−1) times to generate the number n of angle-variableintersecting microbeam arrays, wherein the number n of angle-variableintersecting microbeam arrays intersect substantially only within theinjury site, the injury site including a marginal volume surroundinginjured tissue.
 5. The method of claim 4, wherein adjacentangle-variable intersecting arrays are separated by a displacementangle, said angularly displacing comprising angularly displacing by anon-zero integer multiple of the displacement angle.
 6. The method ofclaim 5, wherein the displacement angle is substantially equal toθ/(n−1), wherein θ is predetermined by an angular access of an X-raysource generating the angle-variable intersecting microbeam arrays tothe injury site, θ being a total angular spread encompassing theangle-variable intersecting microbeam arrays.
 7. The method of claim 6,wherein θ is substantially in a range of about 130 degrees to about 150degrees.
 8. The method of claim 3, wherein said generating comprisesgenerating the angle-variable intersecting microbeam arrays for one of ahorizontal, vertical, and slanted irradiation orientation of the atleast two parallel, spatially distinct microbeams.
 9. The method ofclaim 8, further comprising additionally generating a second number n ofangle-variable intersecting microbeams arrays for another one of ahorizontal, vertical and slanted irradiation orientation of the at leasttwo parallel, spatially distinct microbeams, for a total number 2n ofsessions, each session being separated by the time interval.
 10. Themethod of claim 9, said additionally generating further comprising oneof reorientating and replacing a multislit collimator between an X-raysource and the subject to change the irradiation orientation.
 11. Themethod of claim 9, wherein the total number 2n of sessions is within arange of from three (3) to thirty (30) sessions.
 12. The method of claim2, wherein the time interval is substantially within a range of fromabout twelve (12) hours to about seven (7) days.
 13. The method of claim1, wherein the subject is positioned in one of an upright position, aside-reclined, and a slanted position, and wherein the at least onearray is directed onto a subject's back, the at least one array beingcentered around a 90-degree angle of incidence.
 14. The method of claim1, wherein the at least one array comprises a center-to-center spacingbetween adjacent microbeams and a thickness of each of the at least twoparallel, spatially distinct microbeams, wherein a ratio of thecenter-to-center spacing to the thickness is substantially in a range ofabout 4 to about
 16. 15. The method of claim 1, wherein each of the atleast two parallel, spatially distinct microbeams comprise a thicknesssubstantially in a range of from about 0.02 mm to 1.0 mm.
 16. The methodof claim 1, wherein said irradiating further comprises generating saidX-ray radiation with an X-ray bremsstrahlung source.
 17. The method ofclaim 16, wherein each of the at least two parallel, spatially distinctmicrobeams comprise a thickness substantially in a range of from about0.1 millimeters to 1.0 millimeter.
 18. The method of claim 1, whereinsaid irradiating comprises generating X-ray synchrotron radiation, eachof the at least two parallel, spatially distinct microbeams comprising abeam thickness substantially in a range of about 20 micrometers to about100 micrometers.
 19. The method of claim 1, wherein said irradiatingfurther comprises generating X-ray radiation having a filtered broadbeam energy spectrum, a half-power energy being substantially in a rangefrom at least about 100 keV to about 250 keV.
 20. The method of claim 1,wherein the therapeutic dose comprises an in-beam in-depth dose in eachmicrobeam substantially in a range from about 30 Gy to about 500 Gy. 21.The method of claim 1, further comprising delivering stem cells to theinjury site.
 22. The method of claim 1, said irradiating furthercomprising delivering said at least one array in a plurality oftemporally discrete pulses of said X-ray radiation.
 23. The method ofclaim 22, wherein the plurality of temporally discrete pulses aresubstantially synchronized with a physiomechanical cycle of the subject.24. The method of claim 23, wherein the physiomechanical cycle comprisesat least one of a cardiac cycle and a cardiopulmonary cycle.